Infection of the cornea with herpes simplex virus (HSV) initiates an innate immune response that plays a major role in limiting virus spread. Production of interferon (IFN) and IFN-induced proteins is an important component of this innate response. Some of the IFN-inducible proteins localize to nuclear structures termed ND1Os, which are the sites of viral DNA localization and initiation of viral transcription. The aims of this grant focus on determining the mechanism of action of two of these IFN-induced proteins, PML and SP100, and the regulation of their expression in human cornea (HCS) cells. Each protein exists in multiple forms as a result of variable mRNA splicing. Our studies show that SP100B is a potent inhibitor of two HSV transcriptional transactivating proteins, VP16 and ICP4. The specificity of SP100B's inhibitory activity will be characterized by examining the action against basal expression and transactivation of a number of viral and cellular promoters. Using the yeast two-hybrid system we will identify cellular protein(s) that function as mediators between SP100B and ICP4. The region of SP100B that confers repression is within a 29 amino acid domain that has the potential to be highly phosphorylated. This region will be mapped to determine whether specific amino acid phosphorylation is essential for activity. We will determine whether localization to the ND1O structure and transcriptional repressive activity are linked. The second protein to be examined is PML, a structural component of ND1Os. Two forms of PML will be evaluated alone and combined with SP100B for their contribution to the transcriptional regulation of HSV genes. The interaction of PML with the viral general transcriptional transactivator ICPO, a protein known to disrupt ND1s, will be characterized by transcription assays in transiently and stably transfected cells and by microscopic localization of green fluorescent protein-tagged forms of PML. HCS cells appear to contain high levels of PML localized to ND1Os, potentially acting to provide an elevated innate immune response. Monoclonal antibodies specific for forms of PML and SP100 will be prepared and used to identify the forms of PML and SP100 naturally present in these corneal cells and determine the changes that occur in their expression in response to IFN treatment and virus infection. These studies will characterize a potent transcriptional repressive mechanism we have identified and determine its contribution to corneal inhibition of HSV replication. This knowledge may be utilized to enhance this innate antiviral response and thereby limit initial and recurrent viral infections in the eye.